Fig 5.

Depletion of clathrin light-chain and clathrin heavy-chain proteins has no effect on JEV infection of neuronal cells. (A) Neuro2a cells were transfected with either GFP-Retro Q shRNA plasmid vector alone (left panel) or GFP-CLC shRNA (right panel) and 72 h later were given a 10-min pulse of Alexa 647-Tf (pseudocolored in red). Note that cells expressing vector alone show efficient Tf uptake, while cells expressing CLC shRNA show a block in Tf endocytosis. Bar, 5 μm. (B) Quantitation of Tf uptake was done as described in Materials and Methods. (C) Western blots showing depletion of CLC in Neuro2a and Vero cells transfected with CLCshRNA plasmid for 72 h and loading control actin. (D) Neuro2a and Vero cells were transfected with shRNA plasmids directed against clathrin light chain (CLC) or control mock plasmid. After 72 h, cells were infected with JEV (MOI, 1). Cells were scored at 24 hpi by immunofluorescence staining of JEV E antigen. Infection was quantified as described above. (E) JEV titers in Neuro2a and Vero cells transfected with control and CLCshRNA plasmids, estimated at 24 hpi (MOI, 1). (F) Western blot showing depletion of CHC in Neuro2a cells transfected with shRNA plasmids for 72 h and loading control actin. (G) JEV infection in Neuro2a cells in the background of CHC depletion was scored as described above. (H) Virus titers (mean ± SD) in culture supernatants of Neuro2a cells depleted of CHC, infected at an MOI of 1, were calculated at 24 hpi by plaque assays. Student's t test was used to generate P values. **, P < 0.01; *, P < 0.05.